摘要
目的获得粉尘螨长链脂肪酸转运蛋白(long-chain fatty acid transport protein,FATP)编码基因及其分子特征。方法根据粉尘螨转录组基因测序结果,获得FATP编码序列片段,据此设计引物,用反转录-聚合酶链式反应从粉尘螨总RNA中扩增全长基因片段,构建原核表达质粒pET28a(+)-FATP。成功获取FATP序列后,利用Expasy、SingaIP 5.0、GOR4和TMpred等软件进行氨基酸序列结构和功能分析,最后利用Clustal Omega及MEGA-X软件进行同源性分析。结果基因编码序列全长1071 bp,编码的蛋白质由356个氨基酸组成,其亲水性指数为-0.39,此蛋白为可溶性蛋白,结构稳定,二级结构包括α-螺旋(25.56%)、延伸主链(23.04%)和无规则卷曲(51.40%)。该蛋白存在两处较明显的跨膜区段,由外向内的跨膜区位于1~20位氨基酸,由内向外的跨膜区在100~116位氨基酸。同源氨基酸序列构建的分子进化树中发现与家蚕聚成一簇。结论获得了粉尘螨FATP编码基因全长及其分子特征,为探讨粉尘螨生理现象、开发粉尘螨控制措施奠定基础。
Objective To identify the gene encoding the long-chain fatty acid transport protein(FATP)of Dermatophagoides farinae and its molecular characteristics.Methods According to the transcriptome sequencing data of D.farinae,we obtained the FATP-coding sequence for designing primers.Then RT-PCR was used to amplify the full-length gene fragment from total RNA of D.farinae,followed by construction of the prokaryotic expression plasmid pET28a(+)-FATP.After obtaining FATP sequence successfully,the structure and function of amino acid sequence were analyzed by using the software of Expasy、singaIP5.0、GOR4 and TMpred,and performed homology analysis through Clustal Omega and MEGAX.Results The full length of the gene coding sequence was 1071 bp.The encoded protein was composed of 356 amino acids.It was soluble,with a grand average of hydropathicity of-0.39,and had a stable structure.The secondary structure included alpha-helix(25.56%),extended strand(23.04%),and random coil(51.40%).The protein had two transmembrane regions,The transmembrane area from the outside to the inside is 1-20 aa,and the transmembrane area from the inside to the outside is 100-116 aa.The evolutionary tree derived from homologous amino acid sequences showed that the sequence of D.farinae was clustered with that of silkworm.Conclusion The full length of the FATP-coding gene and the molecular characteristics of FATP are identified in D.farinae,laying a foundation for exploring its physiological phenomena and developing control measures.
作者
朱晗婷
吴美丽
周鹰
吴冰
崔玉宝
ZHU Han-ting;WU Mei-li;ZHOU Ying;WU Bing;CUI Yu-bao(The Affiliated Wuxi People’s Hospital of Nanjing Medical University,Wuxi 214023,Jiangsu Province,China;Wuxi Children’s Hospital)
出处
《中国媒介生物学及控制杂志》
CAS
2020年第2期169-174,共6页
Chinese Journal of Vector Biology and Control
基金
国家自然科学基金(31572319,31272369)
无锡市卫生计生科研重大项目(Z201701)
江苏省第五期“333工程”科研项目(BRA2017216)
江苏省重点研发计划(BE2018627)。
关键词
粉尘螨
长链脂肪酸转运蛋白
分子克隆
生物信息学
Dermatophagoides farinae
Long-chain fatty acid transport protein
Molecular cloning
Bioinformatics
